Cytology meets Molecular: Don t throw away your microscope! British Association for Cytopathology November 4, 2017

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1 Cytology meets Molecular: Don t throw away your microscope! British Association for Cytopathology November 4, 2017 Andrew Fischer, M.D. Director of Cytopathology University of Massachusetts

2 Theme: How will molecular testing and cytology evolve What can molecular testing replace, using next generation sequencing and bladder cancer screening as an example. What molecular testing cannot do. The criteria of malignancy, when viewed from the proper perspective, provide an irreplaceable insight into the function of oncogenes. New microscopy techniques fluorescent optical sectioning and live cell imaging will keep our cytomorphologic skills in demand.

3 High grade urothelial carcinoma, microbiopsy cytology sample from catheterized ureter Cost in US (medicare): ~40$ Sensitivity for high grade UC: Up to about 90%* Sensitivity for ALL UC, high and low grade, <50% Lee et al, Causes of False-Negative for High-Grade Urothelial Carcinoma in Urine Cytology. Diagn. Cytopathol. 2016;44:

4 Cost of Next generation sequencing: As low as 15,000 base pairs per penny 3 billion basepairs in whole human genome Only 1.5% is exomic Need 30X coverage (average 30 reads per DNA molecule) Costs approaching $1000 for biggest machines. = ~15,000 bp for 1 cent

5 Next generation sequencing for urothelial carcinoma screening Compared to germline sequencing (two alleles, so 50% of sequences are from one allele) NGS needs many individual reads of the sequence to detect tumors that are diluted with many normal gene sequences. Many genes are potentially mutated in cancers, so you need to sequence many segments of DNA to have a high sensitivity (~600 segments, ~100,000 bp). Would cost (best case scenario) at least $150. A DNA methylation survey identified only ~150 DNA segments, preferentially methylated in cancer, that may be more suitable.

6 For NGS to work, need a high proportion of abnormal cells. NGS could not be effective for this case. In general, most molecular tests cannot work if the input material is not assessed. Cytology is ideal for assessing input material, because so few cells (~5000) are needed.

7 UroMark NGS platform for detecting high and low grade bladder cancers Detected bladder cancer with sensitivity of 98%, specificity of 97%. To detect 1% tumor cells, would need very roughly 2000 reads for each amplicon (20 individual tumor DNA sequences). Assume each amplified sequence is 100 base pairs X 150 X 100 / 15,000 = $20.00

8 Will Molecular take over urine cytology? NGS-based testing is poised to replace at least some urine cytology as a screening test for cancer. Likely to evolve through a phase of reflex testing for cytologic atypia when percent abnormal cells is high. Most likely to first replace cytology for follow-up of bladder cancer patients with known mutations.

9 96.5 % of driver genes for PTC have been identified.

10 TCGA has not shed ANY light on why PTC has diagnostic changes in chromatin and nuclear shape. Does Cytomorphology matter? Molecular vs. Cytology MAP kinase pathway, From Wikipedia, accessed

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12 Structure of the nucleosome, showing ~150 nucleotides of DNA wrapped around the 4 pairs of histones. Note the protruding histone tails. From Luger et al., Nature 389:251, 1997

13 A structure-function relation should also exist at the cellular level Normal ductal cells vs pancreatic IPMN

14 Hypothetical phylogeny of a cancer Fischer, Young, DeLellis, J Cellular Biochem 93:28-36, 2004

15 Hallmarks of cancer are the hypotheses for how oncogenes work. In fact, 35 years after the discovery of oncogenes, we still do not know how they actually work! Hanahan and Weinberg, Cell 100:57, 2000 Hanahan and Weinberg, Cell 144:646, 2011

16 Phylogeny of Darwin s Finches Fischer, Young, DeLellis, J Cellular Biochem 93:28-36, 2004

17 Darwin knew that the morphologic features that distinguish related species are ultimately caused by the heritable elements (genes) that differ between the species. relate in the most essential yet often hidden manner to the mechanism of increased fitness.

18 Darwin s theory predicts there should be a relation between the morphologic changes diagnostic of cancers, the genes that are active in the cancers, and (most importantly) the functional changes that allow this cellular evolution to take place

19 ASC-sponsored Classification of the Criteria of Malignancy 1. Tissue-level architectural changes reflecting clonal expansion. 2. Changes reflecting genetic instability 3. Sub-cellular changes, unrelated to genetic instability, conferring increased cellular fitness A. Directly induced by oncogenes B. Likely heritable, but no known genetic basis Fischer et al., J Cellular Biochem 2010, 110:75-811

20 Group 1: Tissue architectural criteria Three common mechanisms for early clonal expansion of epithelial cells Crowding Papillary formation True stratification Loss of contact inhibition, Pseudostatification Anchorage-independent growth, resistance to anoikis Fischer, Fundamentals of Cytological Diagnosis and Its Biological Basis. In: Pathobiology of Human Disease. Published by Elsevier, p

21 Normal ductal cells Columnar cell change breast

22 Papillary thyroid carcinoma (Photomicrographs courtesy of Manon Auger, McGill)

23 Hepatocellular carcinoma, defined by true stratification away from endothelial cells. CD 34 staining to show endothelial cells

24 The development of true stratification typically coincides with loss of ( basalapical ) polarity. Stratified cells can then orienting freely toward any surface (cribriforming). There is increasing evidence for involvement of oncogenes in breaking down normal basal-apical polarity

25 Mammary carcinoma, with randomized positioning of secretions and random nuclear axis. Objective markers of basal-apical polarity would likely be useful for diagnosis

26 Group 2: Cytologic changes reflecting genetic instability Cell to cell variation in: Total DNA content (total integrated amount of hematoxylin staining), reflecting chromosome instability Cytoplasmic features (evidence of phenotypic instability) Chromatin packaging patterns (?Epigenetic instability?)

27 DNA content variation reflecting chromosomal instability in two HSIL pap tests

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29 Polyploidization is a common benign change

30 Benign polyploidization compared to malignant nuclear pleomorphism

31 Group 3: Cell structural changes, not related to genetic instability* Nuclear shape abnormalities Intermediate filament organization Other cytoplasmic diagnostic changes Abnormal nucleolar prominence Chromatin alterations *Several of these criteria are known to be directly induced by the cancer genes active in the tumor

32 Group 3: Intermediate filament abnormalities E4 gene of HPV mediates intermediate filament collapse

33 Group 3: Collapse of intermediate filaments (keratin 20) in Merkel cell (small cell) carcinoma

34 Normal prostate epithelium Fischer et al. (J Cellular Biochem, 2004) Prostatic intraepithelial neoplasia: Diagnostic feature is nucleolar prominence without reactive cytoplasm

35 Papillary thyroid carcinoma

36 Papillary thyroid carcinoma compared to normal thyroid

37 Thyroid model of carcinogenesis Follicular adenoma Papillary thyroid carcinoma H-RAS et al. RET/PTC TRK/PTC B-RAF Normal thyroid epithelium

38 Ret/PTC expressed in normal human thyroid cells Fischer et al, Am J Pathol 153:1443, 1998

39 Normal human thyroid epithelium

40 Human thyroid epithelium expressing RET Tyrosine kinase

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42 Human thyroid epithelium expressing H(V14)- RAS

43 Active RET/PTC Inactive RET/PTC (control) Nuclear lamina irregularity is induced by RET/PTC in interphase. Cells were microinjected with RET/PTC 6 hours previously and stained for lamins (green) and RET (red) Fischer et al., Am J Pathol 163:1091, 2003

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45 Waggle dance of the honeybee: A distinguishing trait from closest relative the less social bumble bees* Different dance dialects have a genetic basis, shown by back-crossing to be defined by a single locus* Likely to be a key determinant of the fitness difference that allowed their evolutionary split. Essentially impossible to decipher this biology based on just the DNA sequence, and impossible to decipher with just snap shot images. *R. N. Johnson, B. P. Oldroyd, A. B. Barron, and R. H. Crozier, J Heredity, 93:170-3, 2002

46 Two photon microscopy Right top: pseudocolored DAPI and fluoresceein to look like H&E. Bottom right: H&E stained paraffin section. From Dan Schmolze et al., Arch Pathol Lab Med, 135:255, 2011.

47 Living human papillary thyroid carcinoma from thyroidectomy specimen. Vibratome sectioned at 300 micron thickness. Stained for 5 minutes with 0.01% acriflavine. 800 nm 2 photon excitation. Optically sectioned about 60 microns in depth. Viewed in just one time point. Diagnostic features of PTC are seen in LIVING CELLS

48 Ending Comments Molecular diagnosis will replace some cytologic tests. However, it will mostly increase the role for cytology, through an increased use of minimally sized biopsies that are ideal when molecular and cytology are used together. Oncogenes are selected for at the cellular level. The DNA level of biology CANNOT by itself inform us about how oncogenes actually work. An increasingly refined understanding of the cytologic criteria of malignancy brings us closer and closer to a description of the exact functional changes in cancer cells. New microscopy techniques make it feasible to visualize new types of diagnostic cellular dynamics, dynamics that will require our expertise for use in diagnosis and prognostication for generations to come.